Methods and compositions for multiple displacement amplification of nucleic acids

ABSTRACT

Disclosed are methods for multiple displacement amplification of a nucleic acid sequence in a sample. The nucleic acid is contacted with a reaction mixture that includes a set of oligonucleotide primers and a plurality of polymerase enzymes. The reaction mixture is subjected to conditions under which the nucleic acid sequence is amplified to produce an amplification product in a multiple displacement amplification reaction. Also disclosed are kits containing a set of oligonucleotide primers with random sequences having lengths of 6 to 8 nucleobases. At least some of the individual members of the primers have one or more ribose modifications that stabilize or lock the ribose ring in a 3′-endo conformation. At least some of the primers have one or more universal nucleobases.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under FBIcontract J-FBI-05-027, FBI contract J-FBI-03-134, and HSARPA contractW81XWH-05-C-0116. The United States Government has certain rights in theinvention.

SUPPORT FIELD OF THE INVENTION

The methods disclosed herein relate to methods and compositions foramplifying nucleic acid sequences.

BACKGROUND OF THE INVENTION

In many fields of research such as genetic diagnosis or forensicinvestigations, the scarcity of genomic DNA can be a severely limitingfactor on the type and quantity of genetic tests that can be performedon a sample. One approach designed to overcome this problem is wholegenome amplification. The objective is to amplify a limited DNA samplein a non-specific manner in order to generate a new sample that isindistinguishable from the original but with a higher DNA concentration.The aim of a typical whole genome amplification technique would be toamplify a sample up to a microgram level while respecting the originalsequence representation.

The first whole genome amplification methods were described in 1992, andwere based on the principles of the polymerase chain reaction (PCR).Zhang and coworkers (Zhang et al. Proc. Natl. Acad. Sci. U.S.A. 1992,89, 5847-5851) developed the primer extension PCR technique (PEP) andTelenius and collaborators (Telenius et al., Genomics 1992, 13, 718-725)designed the degenerate oligonucleotide-primed PCR method (DOP-PCR).

PEP involves a high number of PCR cycles; using Taq polymerase and 15base random primers that anneal at a low stringency temperature.Although the PEP protocol has been improved in different ways, it stillresults in incomplete genome coverage, failing to amplify certainsequences such as repeats. Failure to prime and amplify regionscontaining repeats may lead to incomplete representation of a wholegenome because consistent primer coverage across the length of thegenome provides for optimal representation of the genome. This methodalso has limited efficiency on very small samples (such as singlecells). Moreover, the use of Taq polymerase implies that the maximalproduct length is about 3 kb.

DOP-PCR is a method which uses Taq polymerase and semi-degenerateoligonucleotides that bind at a low annealing temperature atapproximately one million sites within the human genome. The firstcycles are followed by a large number of cycles with a higher annealingtemperature, allowing only for the amplification of the fragments thatwere tagged in the first step. This leads to incomplete representationof a whole genome. DOP-PCR generates, like PEP, fragments that are inaverage 400-500 bp, with a maximum size of 3 kb, although fragments upto 10 kb have been reported. On the other hand, as noted for PEP, a lowinput of genomic DNA (less than 1 ng) decreases the fidelity and thegenome coverage (Kittler et al. Anal. Biochem. 2002, 300, 237-244).

Multiple displacement amplification (MDA, also known as stranddisplacement amplification; SDA) is a non-PCR-based isothermal methodbased on the annealing of random hexamers to denatured DNA, followed bystrand-displacement synthesis at constant temperature (Blanco et al. J.Biol. Chem. 1989, 264, 8935-8940). It has been applied to small genomicDNA samples, leading to the synthesis of high molecular weight DNA withlimited sequence representation bias (Lizardi et al. Nature Genetics1998, 19, 225-232; Dean et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99,5261-5266). As DNA is synthesized by strand displacement, a graduallyincreasing number of priming events occur, forming a network ofhyper-branched DNA structures. The reaction can be catalyzed by thePhi29 DNA polymerase or by the large fragment of the Bst DNA polymerase.The Phi29 DNA polymerase possesses a proofreading activity resulting inerror rates 100 times lower than the Taq polymerase (Lasken et al.Trends Biotech. 2003, 21, 531-535).

The methods described above generally do not successfully amplify DNAsamples when the quantity of template DNA being amplified is below thelevel of one 1 nanogram (ng). Problems encountered during suchamplification attempts include, for example, poor representation of theoriginal template DNA in the amplified product (Dean et al. Proc. Natl.Acad. Sci U.S.A. 2002, 99, 5261-5266) and competing amplification ofnon-template DNA (Lage et al. Genome Research 2003, 13, 294-307).

There remains a long felt need for methods and kits for performing wholegenome amplification reactions on small quantities of DNA. The presentinvention satisfies this need.

SUMMARY OF THE INVENTION

Disclosed are methods for multiple displacement amplification of anucleic acid sequence in a sample. The nucleic acid is contacted with areaction mixture that includes a set of oligonucleotide primers and aplurality of polymerase enzymes. The reaction mixture is subjected toconditions under which the nucleic acid sequence is amplified to producean amplification product in a multiple displacement amplificationreaction.

Also disclosed are reaction mixtures containing a plurality ofpolymerase enzymes, a set of natural deoxynucleotide triphosphates and aplurality of compatible solutes.

Also disclosed are kits containing a set of oligonucleotide primers withrandom sequences having lengths of 6 to 8 nucleobases. At least some ofthe individual members of the primers have one or more ribosemodifications that stabilize or lock the ribose ring in a 3′-endoconformation. At least some of the primers have one or more universalnucleobases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the number of allele calls made in analysis of ahuman DNA sample using three different amplification mixtures and anon-amplified control mixture.

FIG. 2 is a plot of the average allelic ratio (log base 2) for allelecalls made in analysis of a human DNA sample using three differentamplification mixtures and a non-amplified control mixture.

FIG. 3 is a plot indicating the quantity of amplification productobtained using three different amplification mixtures.

FIG. 4 is an agarose gel photo of DNA products obtained using threedifferent amplification mixtures.

DEFINITIONS

To facilitate an understanding of the methods disclosed herein, a numberof terms and phrases are defined below:

The term “allele” as used herein, is any one of a number of viable DNAcodings occupying a given locus (position) on a chromosome. Usuallyalleles are DNA (deoxyribonucleic acid) sequences that code for a gene,but sometimes the term is used to refer to a non-gene sequence. Anindividual's genotype for that gene is the set of alleles it happens topossess. In a diploid organism, one that has two copies of eachchromosome, two alleles make up the individual's genotype.

The term “allelic balance” as used herein, refers to the ratio of thequantity of the minor allele to the quantity of the major allele.

The term “allele call” as used herein, refers to successfulcharacterization of an allele by a given analysis method. If theanalysis provides successful characterization of both alleles of a genelocus of a DNA sample, it is said that two allele calls are made. If oneallele is characterized while the other allele is not characterized, itis said that one allele call is made. If neither of the two alleles issuccessfully characterized, no allele calls are made.

The term “amplification,” as used herein, refers to a process ofmultiplying an original quantity of a nucleic acid template in order toobtain greater quantities of the original nucleic acid.

The term “compatible solute” as used herein, refers to a class ofcompounds that stabilize cells and cellular components. Compatiblesolutes include, for example, amino acids and their derivatives, andcarbohydrates.

The term “genome,” as used herein, generally refers to the complete setof genetic information in the form of one or more nucleic acidsequences, including text or in silico versions thereof. A genome mayinclude either DNA or RNA, depending upon its organism of origin. Mostorganisms have DNA genomes while some viruses have RNA genomes. As usedherein, the term “genome” need not comprise the complete set of geneticinformation.

The term “hexamer” as used herein refers to a polymer composed of sixunits. More specifically, the term hexamer is used to describe anoligonucleotide primer having six nucleotide residues.

The term “heptamer” as used herein refers to a polymer composed of sevenunits. More specifically, the term heptamer is used to describe anoligonucleotide primer having seven nucleotide residues.

The term “hybridization,” as used herein refers to the process ofjoining two complementary strands of DNA or one each of DNA and RNA toform a double-stranded molecule through Watson and Crick base-pairing orpairing of a universal nucleobase with one of the four naturalnucleobases of DNA (adenine, guanine, thymine and cytosine).

The term “locked nucleic acid” (LNA), refers to a modified RNAnucleotide. The ribose moiety of a locked nucleotide is modified with anextra bridge connecting 2′ and 4′ carbons. The ribose structure withthis bridge is a bicyclic structure. The bridge “locks” the ribose in a3′-endo structural conformation, which is often found in the A-form ofDNA or RNA. LNA nucleotides can be mixed with DNA or RNA bases in anoligonucleotide. The locked ribose conformation enhances base stackingand backbone pre-organization and has the effect of significantlyincreasing the thermal stability (melting temperature) of a DNA duplex.Two examples of LNAs are the classic LNA which has a single carbon(methylene) bridge between the ribose 2′ and 4′ carbons and another typeof LNA known as ENA, which has an ethylene bridge between the ribose 2′and 4′ carbons. An individual LNA nucleotide is considered to be anexample of a modified nucleotide which can be incorporated into DNA andoligonucleotide primers.

The term “multiple displacement amplification” as used herein, refers toa non-PCR-based isothermal method based on the annealing of randomhexamers to denatured DNA, followed by strand-displacement synthesis atconstant temperature. It has been applied to small genomic DNA samples,leading to the synthesis of high molecular weight DNA with limitedsequence representation bias. As DNA is synthesized by stranddisplacement, a gradually increasing number of priming events occur,forming a network of hyper-branched DNA structures. The reaction can becatalyzed by the Phi29 DNA polymerase or by the large fragment of theBst DNA polymerase.

The term “nucleic acid” as used herein, refers to ahigh-molecular-weight biochemical macromolecule composed of nucleotidechains that convey genetic information. The most common nucleic acidsare deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The monomersfrom which nucleic acids are constructed are called nucleotides. Eachnucleotide consists of three components: a nitrogenous heterocyclicbase, either a purine or a pyrimidine (also known as a nucleobase); anda pentose sugar. Different nucleic acid types differ in the structure ofthe sugar in their nucleotides; DNA contains 2-deoxyribose while RNAcontains ribose.

The term “nucleobase” as used herein, refers to a nitrogenousheterocyclic base, either a purine or a pyrimidine in a nucleotideresidue or within a nucleic acid. The term nucleobase is used herein todescribe the length of a given oligonucleotide primer according to thenumber of nucleotide residues included in the oligonucleotide primer.

The term “octamer” as used herein refers to a polymer composed of eightunits. More specifically, the term octamer is used herein to describe aprimer having eight nucleotide residues.

The term “polymerase” as used herein, refers to an enzyme that catalyzesthe process of replication of nucleic acids. More specifically, DNApolymerase catalyzes the polymerization of deoxyribonucleotidesalongside a DNA strand, which the DNA polymerase “reads” and uses as atemplate. The newly-polymerized molecule is complementary to thetemplate strand and identical to the template's partner strand.

The term “primer,” as used herein refers to an isolated oligonucleotidewhich is capable of acting as a point of initiation of synthesis whenplaced under conditions in which synthesis of a primer extension productwhich is complementary to a nucleic acid strand is induced, (i.e., inthe presence of nucleotides and an inducing agent such as DNA polymeraseand at a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer, use ofthe method, and the parameters used for primer design, as disclosedherein.

The term “processivity,” as used herein, refers to the ability of anenzyme to repetitively continue its catalytic function withoutdissociating from its substrate. For example, Phi29 polymerase is ahighly processive polymerase due to its tight binding of the templateDNA substrate.

The term “Profiler” as used herein, is a generic term that refers to anassay used to characterize a group of genetic loci. Any group of geneticloci may be analyzed. One of the more common groups of loci generallyincludes a core group of about 13 STR marker loci known as the CODISgroup (Combined DNA Index system). In a given Profiler assay, the STRsare characterized by a capillary electrophoresis method that detectsfluorescently tagged STR amplification products. An example of aspecific profiler assay is the AmpFlSTR® Profiler Plus™ assay (AppliedBiosystems, Foster City, Calif.).

The term “quality of amplification” refers collectively to the yield (orfold amplification) of amplified nucleic acid, the specificity ofamplification with respect to non-template nucleic acids, and theperformance of the resulting amplified products in the Profiler™ assay.

The term “reaction mixture” as used herein refers to a mixturecontaining sufficient components to carry out an amplification reaction.

The term “representation” as used herein, refers to a measure ofretaining the original characteristics of the template DNA beingamplified in the multiple displacement amplification reaction. Forexample, if the template DNA is from an individual who has an allelicbalance of 1.5 to 1.0 at a particular genetic locus, and the amplifiedDNA indicates that the allelic balance is 2.0 to 1.0 at that same locus,one would conclude that the amplification reaction resulted in poorrepresentation relative to a different amplification reaction producingan allelic balance of 1.6 to 1.0 for the same template DNA sample.

The term “sensitivity” as used herein refers to a measure of the abilityof a given reaction mixture to amplify very low quantities of DNA suchas, for example, quantities in the picogram range. For example, a givenreaction mixture that produces a useful quantity of a amplified DNA inan amplification reaction starting from a given quantity of template DNAis more sensitive than another given reaction mixture which cannotproduce a useful quantity of DNA from the same quantity of DNA.

The term “set of oligonucleotide primers” as used herein, refers to aplurality of oligonucleotide primers whose members are not matched up inforward and reverse pairs as they generally are for a typical polymerasechain reaction. As used herein a “set of oligonucleotide primers” is aplurality of oligonucleotide primers having generally random sequencesand ranging in length from about six to about eight nucleobases.

The term “short tandem repeat” or “STR” as used herein refers to a classof polymorphisms that occurs when a pattern of two or more nucleotidesare repeated and the repeated sequences are directly adjacent to eachother. The pattern can range in length from 2 to 10 base pairs (bp) (forexample (CATG)n in a genomic region) and is typically in the non-codingintron region. By examining several STR loci and counting how manyrepeats of a specific STR sequence there are at a given locus, it ispossible to create a unique genetic profile of an individual.

The term “template nucleic acid” or “template DNA” as used herein,refers to the strand or strands of DNA that are replicated in anamplification reaction catalyzed by a polymerase enzyme. Morespecifically, a template nucleic acid represents the target nucleic acidadded to the amplification reaction. The target nucleic acid refers tothe object of analysis. A sample containing the template nucleic acidmay contain other nucleic acid contaminants which would not be equallyconsidered as template nucleic acids. For example, if the objective ofanalysis is to identify an individual from his or her DNA, a samplecontaining this DNA would be obtained and amplified. This DNA is thetarget of analysis and represents the template. Contaminating nucleicacids may be present but are not considered as template DNA because theyare not the object of analysis.

The term “universal nucleobase” as used herein, refers to a nucleobasethat is capable of forming a base pair with any of the four naturalnucleobases of DNA; adenine, guanine, thymine or cytosine.

The term “whole genome amplification” or “WGA” as used herein generallyrefers to a method for amplification of a limited DNA sample in anon-specific manner, in order to generate a new sample that isindistinguishable from the original but with a higher DNA concentration.The ideal whole genome amplification technique would amplify a sample upto a microgram level while maintaining the original sequencerepresentation. The DNA of the sample may include an entire genome or aportion thereof. Degenerate oligonucleotide-primed PCR (DOP), primerextension PCR technique (PEP) and multiple displacement amplification(MDA), are examples of whole genome amplification methods.

Description of Embodiments Overview

Disclosed herein are methods, reaction mixtures, kits and primercompositions for multiple displacement amplification reactionsappropriate for amplifying small quantities of DNA. The amplified DNA isparticularly useful for carrying out human forensics testing and is alsouseful for clinical testing or for identification of pathogens inenvironmental samples.

Methods and Reaction Mixtures

The methods for multiple displacement amplification include the steps ofpreparing a reaction mixture that enhances the sensitivity, allelicbalance and quality of the template DNA being amplified.

Sensitivity is a measure of the ability of the reaction mixture toamplify the smallest quantities of DNA.

Allelic balance refers to the ratio of the quantities of two forms of agiven allele. If representation of the original DNA is maintained in agiven multiple displacement amplification reaction, the allelic balanceshould be maintained. Poor representation of the original template DNAin the amplified product is a common problem associated with wholegenome amplification methods (Dean et al. Proc. Natl. Acad. Sci U.S.A.2002, 99, 5261-5266).

Quality is a general measure of the extent of amplification obtained andthe percentage of the amplification products from the template (or“target”) DNA. A further measure of quality is the performance of theamplification product in a DNA profiler™ assay where specific human DNAmarkers are measured for the purpose of identifying human individuals.

In some embodiments, the reaction mixtures employed in the multipledisplacement amplification reactions are compounds known as compatiblesolutes. These compounds stabilize cells and cellular components whenexposed to extreme conditions. In bacteria, the uptake or synthesis ofcompatible solutes renders the cells and their enzymatic machinery moreresistant to stress-inducing environmental conditions such as highosmolarity or high temperatures. These protective effects can beextended to amplification reactions by inclusion as components of anamplification reaction mixture. The compatible solute betaine(N,N,N-trimethylglycine), is an amino acid that acts as anosmoprotectant which increases the resistance of polymerase enzymes todenaturation and also allows amplification reactions to overcome lowlevels of contaminants that often result in low-quality amplificationreactions (Weissensteiner et al. Biotechniques 1996, 21, 1102-1108). Thecompatible solute trehalose is a non-reducing disaccharide in which twoD-glucose units are linked by an alpha-alpha-1,1-glycosidic bond.Trehalose been identified as an enhancer of the polymerase chainreaction (PCR) acting to lower the melting temperature of DNA andincreasing the thermal stability of Taq polymerase (Speiss et al. Clin.Chem. 2004, 50, 1256-1259).

In some embodiments, the compatible solutes are betaine, trehalose, or acombination thereof. In some embodiments, the concentrations of betaineincluded in the reaction mixtures is in a range between about 0.25 M toabout 1.5 M or any fractional concentration therebetween. In someembodiments, the concentration of betaine is between about 0.75 M toabout 1.0 M and preferably about 0.8 M. In some embodiments, theconcentration of trehalose included in the reaction mixtures is in arange between about 0.2 M to about 1.0 M or any fractional concentrationtherebetween. In some embodiments, the concentration of betaine isbetween about 0.6 M to about 1.0 M and preferably about 0.8 M.

In some embodiments, the reaction mixtures employed for the multipledisplacement amplification include a plurality of polymerase enzymes. Insome embodiments, the catalytic activities include 5′→3′ DNA polymeraseactivity, 3′→5′ exonuclease proofreading activity, and DNA repairactivities such as, for example, 5′→3′ excision repair activity.Examples of various polymerase enzymes include, but are not limited to,the following: Phi29, Klenow fragment, T4 polymerase, T7 polymerase,BstE polymerase, E. coli Pol I, Vent, Deep Vent, Vent exo-, Deep Ventexo-, KOD HiFi, Pfu ultra, Pfu turbo, Pfu native, Pfu exo-, Pfu exo-Cx,Pfu cloned, Proofstart (Qiagen), rTth, Tgo and Tfu Qbio. Thesepolymerases are known and most are commercially available. In apreferred embodiment, said plurality of polymerases is Phi29 and atleast one more polymerase enzyme. More preferably, said plurality ofpolymerases is Phi29 and at least one Pol I polymerase. Most preferably,said plurality of polymerases is Phi29 and E. coli Pol I.

In embodiments wherein said plurality of polymerases in the reactionmixture is Phi29 polymerase, and E coli Polymerase I (also known as PolI), the enzymes include 5′→3′ DNA polymerase activity, 3′→5′ exonucleaseproofreading activity, and 5′→3′ excision repair activity. In someembodiments, Phi29 is the major polymerase while E. coli DNA PolymeraseI is present at lower activity levels. In some embodiments, about 10units of Phi29 polymerase is present in the reaction mixture while about2.0 units of E. coli DNA Polymerase I is present in the reactionmixture.

In other embodiments, other non-polymerase enzymes or accessory proteinsare included in the reaction mixtures such as, for example, helicase,gyrase, T4G32 and SSBP for example. These accessory proteins are knownand most are commercially available.

In some embodiments, the reaction mixture further includespyrophosphatase which serves to convert pyrophosphate to phosphate.Pyrophosphate accumulates in the reaction mixture as a result of theamplification reaction (one equivalent of pyrophosphate is generatedfrom each incorporated deoxynucleotide triphosphate added and is knownto inhibit the amplification reaction). In some embodiments about 0.004units of pyrophosphate is added to the reaction mixture.

In some embodiments, it is preferable that bovine serum albumin (BSA) isnot included in the reaction mixture because commercially obtained lotsof BSA often are contaminated with bovine DNA which represents asignificant contaminant that may be co-amplified with the template DNAin the amplification reaction.

In some embodiments, the amplification reaction is an isothermalamplification reaction, meaning that it is carried out at a constanttemperature. In some embodiments, the reaction conditions includethermal cycling where the temperature of the reaction mixture issuccessively raised and lowered to pre-determined temperatures in orderto melt and anneal the two strands of DNA. In some embodiments, it maybe appropriate to perform an isothermal amplification if, for example,representation is maintained by amplification with Phi29 polymerase. Inother embodiments, a greater contribution of enzymatic activityoriginating from different polymerase (such as Pol I, for example) maybe advantageous, in which case thermal cycling may be included in thereaction conditions.

In some embodiments, the amplification reaction results in theamplification of template DNA of a whole genome, or a substantialportion thereof.

In some embodiments, the multiple displacement amplification reactionproduces an amplification product from a template DNA at a constantratio relative to production of amplification products of otherextraneous DNAs in a given sample. This preserves the representation ofthe original template DNA.

In some embodiments, the total quantity of the template DNA added to thereaction mixture for amplification is at least about 2 picograms.

Primer Sets

The primer sets used in the amplification reactions disclosed herein aregenerally defined as a plurality of oligonucleotide primers. In someembodiments, the primers have random sequences that hybridize randomlyto the template nucleic acid at positions of the template thatsubstantially base-pair with the primers. The members of the primer setsmay be random hexamers (primers with six nucleotide residues), randomheptamers (primers with seven nucleotide residues), or random octomers(primers with eight nucleotide residues). The syntheses of suchhexamers, heptamers and octomers with random sequences are accomplishedby known procedures.

In some embodiments, the primers include modifications that increasetheir affinity for the template nucleic acid. In certain embodiments,the modifications include substituents on the ribose ring of a givennucleotide residue of a given primer, which stabilize or lock the ribosering in the 3′-endo conformation which provides for a higher affinity ofthe nucleotide residue for a pairing nucleotide residue on a templatenucleic acid.

The conformation of the ribose sugar of a nucleotide residue within aprimer is influenced by various factors including substitution at the2′-, 3′- or 4′-positions of the pentofuranosyl sugar. Electronegativesubstituents generally prefer the axial positions, while stericallydemanding substituents generally prefer the equatorial positions(Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element (Gallo et al. Tetrahedron 2001, 57, 5707-5713;Harry-O'kuru et al., J. Org. Chem., 1997, 62), 1754-1759; and Tang etal., J. Org. Chem. 1999, 64, 747-754). Alternatively, preference for the3′-endo conformation can be achieved by deletion of the 2′-OH asexemplified by 2′-deoxy-2′-F-nucleosides (Kawasaki et al., J. Med. Chem.1993, 36, 831-841), which adopts the 3′-endo conformation positioningthe electronegative fluorine atom in the axial position. Othermodifications of the ribose ring, for example substitution at the4′-position to give 4′-F modified nucleosides (Guillerm et al., Bioorg.Medicinal Chem. Lett. 1995, 5, 1455-1460 and Owen et al., J. Org. Chem.1976, 41, 3010-3017), or for example modification to yield methanocarbanucleoside analogs (Jacobson et al., J. Med. Chem. Lett. 2000, 43,2196-2203 and Lee et al., Bioorg. Med. Chem. Lett. 2001, 11, 1333-1337)also induce preference for the 3′-endo conformation.

The most common locked nucleic acid modification is a 2′ to 4′ methylenebridge which locks the ribose ring in the 3′-endo conformation. Thismodification is often abbreviated as “LNA,” meaning “locked nucleicacid. Another type of locked nucleic acid is referred to as ENA(ethylene-bridged nucleic acid). This modification includes a 2′ to 4′ethylene bridge. The synthesis and preparation of the 2′ to 4′ bridgedmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). The 2′ to 4′ bridged monomers and preparation thereof arealso described in WO 98/39352 and WO 99/14226. The first analogs of 2′to 4′ bridged nucleic acids, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of oligodeoxyribonucleotide duplexes with 2′ to4′ bridged nucleoside analogs as substrates for nucleic acid polymeraseshas also been described (WO 99/14226). Furthermore, the synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem. 1998, 63, 10035-10039). In addition,2′-amino- and 2′-methylamino-LNAs have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

In some embodiments, at least some of the primers contain at least one2′ to 4′ bridged nucleotide residues or at least two 2′ to 4′ bridgednucleotide residues. In other embodiments, 2′ to 4′ bridged nucleotideresidues are located at the 2′ and 5^(th) positions of theoligonucleotide primers. These embodiments of the primer sets arehexamers, heptamers or octamers or any combination thereof.

In some embodiments, the primers have random sequences with theexception of having specifically located universal nucleobases such asinosine for example. The specific locations of the inosine nucleobasesare preferably at the two final terminal nucleobases of a giveninosine-containing primer.

In some embodiments, one or more phosphorothioate linkages areincorporated into the primers at the 3′ end of a given primer for thepurpose of making the primer more resistant to nuclease activity.

Primer Kits

Some embodiments also provide kits comprising the primers disclosedherein.

In some embodiments, the kits comprise a sufficient quantity of apolymerase enzyme having high processivity. In some embodiments, thehigh processivity polymerase is Phi29 polymerase or Taq polymerase. Inother embodiments, the high processivity polymerase is a geneticallyengineered polymerase whose processivity is increased relative to thenative polymerase from which it was constructed.

In some embodiments, the kits comprise a sufficient quantity of anadditional polymerase in addition to a high processivity polymerase, forimprovement of the characteristics of the amplification reaction. Insome embodiments, the additional polymerase is E. coli Pol I polymerase.

In some embodiments, the kits comprise a sufficient quantity ofpyrophosphatase which catalyzes conversion of pyrophosphate to twoequivalents of phosphate. Pyrophosphate is known to inhibit polymerasereactions.

In some embodiments, the kits further comprise deoxynucleotidetriphosphates, buffers, and buffer additives such as compatible solutesincluding trehalose and betaine at concentrations optimized for multipledisplacement amplification.

In some embodiments, the kits further comprise instructions for carryingout targeted whole genome amplification reactions.

EXAMPLES Example 1 Effects of Compatible Solutes on Sensitivity ofAmplification Reaction

The purpose of this series of experiments was to investigate the effectsof the compatible solutes trehalose and betaine on enhancement of thesensitivity of the amplification reaction where sensitivity reflects theability to successfully amplify the target DNA at low concentrationsbelow the 1 nanogram level.

Initial investigations indicated that the concentration of trehalosethat provides an optimal increase in sensitivity of the amplificationreaction was approximately 0.8 M, as indicated in an amplificationreaction of 50 picograms of human DNA, followed by a calculation of thepercentage of alleles detected in analysis of the amplified DNA obtainedin the reaction. Likewise, the optimal concentration of betaine wasfound to be approximately 0.8 M.

In order to investigate the effects of compatible solutes on thesensitivity of the amplification reaction, amplification reactionmixtures were developed as indicated in Table 1.

TABLE 1 Amplification Mixtures Final Conc. Mixture 1 Template DNA 5 μlfrom dilution variable to extinction described below. Tris HCl 0.04025MTris Base 0.00975M Magnesium Chloride 0.012M Ammonium Sulfate 0.01M dNTPmix 100 mM (25 mM each) 2 mM each DTT 0.004M Primer Pair Mix 0.05 mMDiluted enzyme in buffer 0.5 units/μl Mixture 2 (0.8M Trehalose)Template DNA 5 μl from dilution variable to extinction described belowTris HCl 0.04025M Tris Base 0.00975M Magnesium Chloride 0.012M AmmoniumSulfate 0.01M Trehalose 0.8M dNTP mix 100 mM (25 mM each) 2 mM each DTT0.004M Primer Pair Mix 0.05 mM Diluted enzyme in buffer 0.5 units/μlMixture 3 (0.8M Trehalose + 0.8M Betaine) Template DNA 5 μl fromdilution variable to extinction described below. Tris HCl 0.04025M TrisBase 0.00975M Magnesium Chloride 0.012M Ammonium Sulfate 0.01M Betaine0.8M Trehalose 0.8M dNTP mix 100 mM (25 mM each) 2 mM each DTT 0.004MPrimer Pair Mix 0.05 mM Diluted enzyme in buffer 0.5 units/μl

FIG. 1 shows the results of a “dilution to extinction” experimentwherein 1 nanogram of human DNA sample SC35495 was successively dilutedand added to the reaction mixtures shown in Table 1 for amplificationprior to analysis of alleles by known methods. The amplificationreactions were carried out as follows: the reaction mixtures of Table 1(1, 2 and 3) were prepared and subjected to the following amplificationconditions over a period of 6 hours in a thermocycler:

1) 30° C. for 4 minutes;

2) 15° C. for 15 seconds;

3) repeat steps 1 and 2 for a total of 150 times;

4) 90° C. for 3 minutes; and

5) hold at 4° C.

The resulting amplified DNA was analyzed for identification of allelesusing a mass spectrometry-based analysis method wherein specific primerpairs are then used to obtain additional specific amplification productsvia PCR of loci. These specific amplification products have lengths upto about 140 nucleobases which are appropriate for base compositionanalysis by mass spectrometry in a manner similar to that disclosed inJiang et al. Clin Chem. 2007, 53, 195-203. FIG. 1 clearly shows thatreaction mixtures 2 and 3 produce amplified DNA from which allele callscan be made at concentration levels as low as 2 picograms.

In another experiment, the resulting amplified DNA was analyzed usingthe Profiler™ fluorescence procedure, for determination of a series ofshort tandem repeat (STR) alleles known as the CODIS (Combined DNA IndexSystem) group. Specific primer pairs were then used to obtain additionalspecific amplification products via PCR of the loci of interest. Theseamplification products were then subjected to the Profiler™ fluorescenceprocedure. In Tables 2 and 3, ten of the thirteen CODIS core STR (shorttandem repeat) loci are included (abbreviations for the loci columnlabels are as follows: D3S=D3S1358; D8S=D8S1179; D21S=D21S11;D185=D18551; D5S=D5S8181; D135=D135317; and D7S=D7S820, while vWAindicates the von Willebrand factor gene; TPDX indicates the thyroidperoxidase gene; FGA indicates the fibrinogen alpha-chain gene; and AMELindicates the amelogenin gene). The leftmost column indicates thequantity of DNA used in the amplification reactions. The numbersappearing under the loci indicate the number of allele calls made in theanalysis according to the Profiler™ method. The objective is to make twoallele calls for as many loci as possible at the lowest possiblequantity of DNA prior to amplification because the ability to do sowould provide the ability to obtain useful forensic DNA samples fromsmall quantities of tissues.

TABLE 2 Sensitivity of Reaction Mixture 1 Quantity of DNA (pg) D3S vWAFGA AMEL D8S D21S D18S D5S D13(H) D7S 1000 2 2 2 2 2 2 2 2 2 2 500 2 2 22 2 2 2 2 2 2 250 2 1 2 1 1 2 1 2 2 2 125 1 2 2 2 2 2 1 2 2 1 63 0 1 0 10 0 1 0 0 0 31 1 1 2 1 0 2 0 0 0 1 16 0 0 0 0 0 0 0 2 0 0 8 0 0 2 0 0 00 0 2 0 4 0 0 0 0 0 0 1 1 0 0 2 0 0 0 0 0 0 0 1 0 0

TABLE 3 Sensitivity of Reaction Mixture 3 Quantity of DNA (pg) D3S vWAFGA AMEL D8S D21S D18S D5S D13(H) D7S 1000 2 2 2 2 2 2 2 2 2 2 500 2 2 22 2 2 2 2 2 2 250 2 2 2 2 2 2 2 2 2 2 125 2 2 2 2 2 2 2 2 2 2 63 2 2 2 21 1 1 2 2 2 31 2 2 1 2 1 2 2 1 2 2 16 2 0 2 1 1 1 1 1 0 0 8 2 2 0 0 0 01 0 0 0 4 2 0 0 0 1 0 0 1 0 1 2 1 0 1 0 1 0 0 0 0 0

Tables 2 and 3 clearly indicate that mixture 3 comprising added solutessubstantially improves sensitivity over that achieved using non-solutemixture 1. Mixture 3, therefore, generates product from trace nucleicacid samples as low as 2 picograms and provides 2 or more allele callsfor trace samples as low as 4 picograms.

Example 2 Effects of Compatible Solutes on Maintaining Allelic Balancein the Amplification Reaction

Allelic balance is a measure that indicates the ratio of quantity ofdetection of the minor allele vs. the major allele. It is desirable tomaintain the allelic balance of a given sample of DNA as it is amplifiedin order to provide an accurate representation of the allelic balance inthe original sample.

A human DNA sample designated SC35495 was amplified according toconditions described in Example 1. In this example, the amounts of thealleles detected were quantified using the commercially available kitQuantifiler™ (Applied Biosystems). The quantity values were converted toLog₂ to provide a more intuitive measure of balance. These values areshown in FIG. 2, where it can be seen that mixture 3 is the best mixturefor maintaining the best representation of the allelic balance. Thisindicates that inclusion of compatible solutes betaine and trehalose tothe reaction mixture improves allelic balance/representation over theamplification reaction over the reaction mixtures without solute or withtrehalose alone.

Example 3 Effects of Compatible Solutes on Maintaining the Quality ofthe Amplification Reaction

The quality of the amplification reaction can be described in terms ofproviding a combined measure of optimal fold amplification, a highpercentage of amplification of the target nucleic acid being analyzed,and optimal performance in the Profiler™ assay.

Shown in FIG. 3 are the results of the determinations of quantity oftemplate DNA amplified by the three reaction mixtures according to theconditions described in Example 1 using 0.1 nanograms of template DNA(panel A) and 1 nanogram of template DNA (panel B). It is clear thatmixture 3 overall produces the most amplified DNA. Furthermore, it wasfound that the 3 mixture produces fewer extraneous non-template peaksdetected in the Quantifier™ assay (not shown) and in 1% agarose gels(FIG. 4) than observed for the other two mixtures. Thus, the inclusionof betaine and trehalose in the reaction mixture significantly improvesthe quality of an amplification reaction over that of mixtures having nosolutes or having trehalose alone.

Example 4 Effects of Inclusion of an Additional DNA Polymerase on theAmplification Reaction

To assess the effect of augmenting the action of Phi29 polymerase,additional polymerase enzymes were individually added to the 3amplification mixture along with 15 picograms of template DNA of humansample SC35495. The samples were amplified as indicated in Example 1.The resulting amplified DNA was analyzed in the Profiler™ assay andallele calls were made and tallied. Table 4 shows the results andindicates that the addition of Pol I polymerase results in an average offour additional allele calls in the experiment and also indicates thataddition of Pol I polymerase is a favorable modification of theamplification mixture.

TABLE 4 Effects of an Additional Polymerase Enzyme on Whole GenomeAmplification as Measured by Allele Calls from Amplified Mixtures.Additional Enzyme Allele Calls Allele Calls Average Included in MixtureExperiment 1 Experiment 2 Allele Calls None 13 12 12.5 Klenow Fragment11 7 9 T4 polymerase 11 9 10 T7 polymerase 14 13 13.5 BstE polymerase 149 11.5 Pol I polymerase 18 15 16.5

The addition of Pol I polymerase further increases the yield ofamplified DNA and also enhances the genotyping of trace amounts of DNA.Addition of a further enzyme, pyrophosphatase is useful becauseaccumulation of pyrophosphate during the amplification process is knownto inhibit polymerase reactions.

Example 5 Design and Testing of Individual Oligonucleotide PrimerModifications

A series of primer motifs were designed for improvement of the quality,sensitivity and balance of the amplification reaction. The modificationsincluded inclusion of inosine nucleobases at specific positions withinthe hexamer, heptamer and octomer primers. Phosphorothioate modifiedlinkages were incorporated into these primers at the two 3′-mostterminal linkages. The most effective placement of inosine nucleobaseswas found to be at the fifth and sixth positions of the hexamer primers,sixth and seventh positions of the heptamer primers and seventh andeighth positions of the octomer primers. These primers containinginosine nucleobases produced less amplified product than thecorresponding primers that did not contain inosine. However, the totalamplified product was found to represent a greater proportion of thetemplate DNA, indicating that inclusion of inosines in the primersimproves the quality of amplification.

The position of the LNA modified nucleotide residues of the heptamerprimers was examined in detail by systematically changing the positionof one or two LNA modifications (L) in random heptamers as indicated inTable 5. The symbols in Table 5 are as follows: N=A, T, C or G;I=inosine; NI=nitroindole; L=LNA (locked versions of A, C, T or G). Inthis experiment, improvements in fold amplification are achieved forprimers having LNA substituted in position 2, position 4, position 5,positions 1 and 4, and positions 2 and 5. LNA substitutions were welltolerated at all positions, with only the most 3′ position showing aslight negative effect (LNA-7). Primers bearing LNA substituted residuesat two positions have a higher fold amplification increase as comparedto those having only a single LNA substituted residue. Moreover,substituting inosine residues at the most 3′ positions of a position 2,position 5 LNA substituted primer further improved fold amplification(LNA-11 and LNA-12). It is notable that the primer containing thenitroindole universal base (NI) did not perform well in theamplification reaction.

TABLE 5 Determination of Optimal Positioning of LNA residues in thePrimers 5′ Primer Nucleotide Residue Position 3′ Fold AmplificationPrimer 1 2 3 4 5 6 7 Relative to Control Control N N N N N N N 1 LNA-1 LN N N N N N 0.9 LNA-2 N L N N N N N 1 LNA-3 N N L N N N N 0.9 LNA-4 N NN L N N N 1.1 LNA-5 N N N N L N N 1.4 LNA-6 N N N N N L N 1 LNA-7 N N NN N N L 0.7 LNA-8 L N N L N N N 7.6 LNA-9 L N N L N N L 0.2 LNA-10 L N NL N I I 4.8 LNA-11 N L N N L I I 9.3 LNA-12 N L N N L N N 4.1 LNA-13 L NN L N NI I None

The preceding examples illustrate that the use of a plurality ofpolymerases, inclusion of compatible solutes, and modifications ofprimers individually and collectively improve the sensitivity ofamplification while preserving representation of the original nucleicacid sample and producing high quality amplification products.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, internet web sites, and the like) cited in thepresent application is incorporated herein by reference in its entirety.Those skilled in the art will appreciate that numerous changes andmodifications may be made to the embodiments of the invention and thatsuch changes and modifications may be made without departing from thespirit of the invention. It is therefore intended that the appendedclaims cover all such equivalent variations as fall within the truescope of the invention.

What is claimed is:
 1. A method for multiple displacement amplificationof a nucleic acid sequence in a sample comprising the steps of:contacting said nucleic acid with a reaction mixture, wherein saidreaction mixture includes a set of oligonucleotide primers and aplurality of polymerase enzymes; and subjecting said reaction mixture toconditions under which said nucleic acid sequence is amplified toproduce an amplified product in a multiple displacement reaction.
 2. Themethod of claim 1 wherein said sample is a forensic sample comprisinghuman DNA.
 3. The method of claim 1 wherein said plurality of polymeraseenzymes have 5′→3′ DNA polymerase activity, 3′→5′ exonuclease activity,and 5′→3′ excision repair activity.
 4. The method of claim 1 whereinmembers of said set of oligonucleotide primers have random sequences. 5.The method of claim 4 wherein said oligonucleotide primers have lengthsof 6, 7 or 8 nucleotide residues.
 6. The method of claim 4 wherein saidoligonucleotide primers include at least one universal nucleobase. 7.The method of claim 6 wherein said universal nucleobase is inosine. 8.The method of claim 6 wherein said universal nucleobase is located atthe 3′-terminal end of said oligonucleotide primers.
 9. The method ofclaim 1 wherein said set of oligonucleotide primers includes primershaving one or more modifications of the ribose ring that favor a 3′-endoconformation of said ribose ring or lock said ribose ring in said3′-endo conformation.
 10. The method of claim 9 wherein saidmodifications comprise a 2′ to 4′ ribose bridge.
 11. The method of claim10 wherein said modifications are located at the 2nd and 5th positionsof said primers.
 12. The method of claim 1 wherein said one or moreenzymes include phi29 polymerase and pol I polymerase.
 13. The method ofclaim 1 wherein said one or more enzymes further includespyrophosphatase.
 14. The method of claim 1 including the proviso thatbovine serum albumin is not included in said reaction mixture.
 15. Themethod of claim 1 wherein said reaction mixture comprises betaine andtrehalose.
 16. The method of claim 1 wherein said conditions comprisethermal cycling of said reaction mixture.
 17. The method of claim 1wherein said multiple displacement amplification results in whole genomeamplification.
 18. The method of claim 1 wherein said multipledisplacement amplification reaction produces said amplification productat a constant ratio relative to production of other amplificationproducts of other nucleic acids present in said sample.
 19. The methodof claim 1 wherein the total quantity of said nucleic acid sequence isbetween about 2 picograms to about 1000 picograms.